短柄ISSR-PCR反应体系的优化

Optimization of the ISSR-PCR reaction system of Quercus glandulifera var.brevipetiolata

以短柄(Quercus glanduliferavar.brevipetiolata)的DNA为材料,对影响PCR扩增效果的一些因素诸如模板DNA用量、Taq酶的用量、镁离子浓度、dNTP的浓度、引物用量和牛血清白蛋白浓度等指标进行筛选和优化。结果显示适合短柄ISSR-PCR分析最适宜的PCR反应条件为:10μL PCR反应体积中,1×Taq酶配套缓冲液(10 mmol.L-1Tris-HCl,pH9.0,50 mmol.L-1KCl,0.1%Triton X-100),0.75UTaq DNA聚合酶,2 mmol.L-1MgCl2,0.2 mmol.L-14×dNTP,12 pmol引物,10 ng模板DNA,2 mg.mL-1牛血清白蛋白。短柄ISSR扩增较适宜的退火温度为54.4℃。

Factors which affect the ISSR-PCR amplification such as template DNA usage, Taq DNA polymerase, Mg^2 ＋ concentration , dNTP concentration, primer usage and bovine serum albumin concentration, were optimized and selected by using the materials of Quercus glandulifera vat. brevipetiolata DNA. The results showed that the conditions suitable for ISSR-PCR of Quercus glandulifera vat. brevipetiolata were as follows： 1×Taq buffer （ 10 mmol·L^-1t Tris-HCl , pH9. 0 , 50 mmol·L^-1 KCl , 0.1% Triton X-100） , 0.75U Taq DNA polymerase,2.0 mmol·L^-1 MgCl2, 0.2 mmol·L^-1 4 ×dNTP, 12 pmol primers, 10ng template DNA, 2 mg·mL^-1 bovine serum albumin. The suitable annealing temperature in the ISSR-PCR reaction system was 54.4℃.